REGULATION OF TRYPANOSOME DNA REPLICATION

动体鞭毛虫代表着一个独特的机会 真核生物DNA复制机制及其协调研究 与细胞周期有关。这些寄生原生动物的区别在于 存在一个含有大约5,000个线粒体的 复制的类似质粒的DNA(微环)连接成一个巨大的 与25-50个拷贝的线粒体DNA(上颌骨)一起形成网络。 动泡体DNA(KDNA)约占细胞总数的25%。 而且，与哺乳动物的线粒体DNA不同，它与 核S阶段。这项提案的长期目标是确定 KDNA复制的生化和酶机制 束锥虫微环的研究及鉴定 小环复制与细胞协调机制的特点 周而复始。运动基质相关蛋白参与启动及可能的作用 小环复制中的预启动步骤将被提纯并用于 补充动丝泡体粗提物，以形成一个微环- 依赖的DNA复制系统。参与RNA引发的酶 小环复制起始点的起始将被提纯并用于 确定起爆机理和起爆部位。抗病毒抗体 纯化的复制蛋白将用于单个蛋白的定位 在动粒体内并确定可能的关联 含有两种拓扑异构酶的额外复制蛋白 在以前的研究中，位于kDNA外围的结构 网络。编码动泡体相关拓扑异构酶的基因将 克隆测序及其RNA和蛋白产物的表达 基因将在细胞周期的不同时间确定。蛋白质- 将寻找拓扑异构酶的蛋白质相互作用，如果发现， 利用来鉴定和纯化可能的相关蛋白 复制设备。 这项建议解决了现代细胞生物学中的基本问题 因此，这与理解正常和不正常有关 细胞和组织的生长和发育。此外，独一无二的功能 运动基质DNA复制机制的研究可能揭示合适的靶点 用于治疗癌症的新型化疗药物的开发 锥虫病和利什曼病。

The kinetoplastid flagellates represent a unique opportunity for investigating eukaryotic DNA replication mechanisms and their coordination with the cell cycle. These parasitic protozoa are distinguished by the presence of a single mitochondrion which contains approximately 5,000 copies of a plasmid-like DNA (minicircles) catenated into an enormous network together with 25-50 copies of the mitochondrial DNA (maxicircles). The kinetoplast DNA (kDNA) accounts for approximately 25% of the cellular DNA and, unlike mammalian mitochndrial DNA, replicates coordinately with the nuclear S phase. The long-term goal of this proposal is to determine the biochemical and enzymatic mechanisms os replication of the kDNA minicircles in the trypanosomatid Crithidia fasciculata and to identify features of the mechanism coordinating minicircle replication with the cell cycle. Kinetoplast-associated proteins involved in initiation and possible pre-initiation steps in minicircle replication will be purified and used to supplement crude kinetoplast extracts in order to develop a minicircle- dependent DNA replication system. The enzymes involved in RNA-primed initiation at minicircle replication origins will be purified and used to determine the mechanism and the sites of initiation. Antibodies against purified replication proteins will be used to localize individual proteins within the kinetoplast and to determine the possible association of additional replication proteins with the two topoisomerase-containing structures located, in previous studies, at the periphery of the kDNA network. The gene encoding the kinetoplast-associated topoisomerase will be cloned and sequenced and the expression of RNA and protein products of the gene will be determined at various times in the cell cycle. Protein- protein interactions of the topoisomerase will be sought and, if found, exploited to identify and purify possible associated proteins of the replication apparatus. This proposal addresses fundamental questions in modern cell biology and therefore is relevant to an understanding of both normal and abnormal growth and development of cells and tissues. In addition, unique features of the kinetoplast DNA replication mechanism may reveal appropriate targets for the development of new chemotherapeutic drugs for the treatment of trypanosomiasis and leishmaniasis.